Immobilization of lipase on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) hydrogel for the synthesis of butyl oleate

ABSTRACT: Lipase from Candida rugosa was immobilized byentrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethylmethacrylate) [poly(VP-co-HEMA)] hydrogel, cross-linked withethylene glycol dimethacrylate (EDMA). The immobilized en-zyme was used in the esterification of oleic acid with butanol inhexane. The activities of the immobilized enzyme preparationsand the leaching of the enzyme from the hydrogel supports withrespect to composition were investigated. The thermal, solvent,and storage stability of the immobilized preparations also weredetermined. Increasing the percentage VP from 0 to 90, whichcorresponds to the increase in the hydrophilicity of the hydrogels,increased the activity of the immobilized enzyme. Lipase immo-bilized onto VP(%):HEMA(%), 90:10 hydrogel had the highest ac-tivity. Increasing the hydrophobicity of the hydrogel (increasingthe percentage HEMA) seemed to decrease leaching of the en-zyme from the support. Immobilized lipase on 100% HEMA hy-drogel indicated highest entrapment and lowest leaching byhexane washing. The lipase immobilized on VP(%):HEMA(%),50:50 hydrogel showed highest thermal, solvent, and storage sta-bility compared to lipase immobilized on other hydrogel compo-sitions as well as the native lipase.

Paper no. J8973 in JAOCS 76, 571–577 (May 1999)

KEY WORDS: Activity, Candida rugosa, entrapment, esterifica-tion, immobilization, lipase, stability.

Enzymes have gained considerable importance as catalysts inorganic synthesis because of their high selectivity undermilder reaction conditions at near ambient temperature andpressure. Furthermore, enzymes are environmentally friendly(1) as they are generally nontoxic and biodegradable. Thus,enzyme-catalyzed reactions have been used for the prepara-tion of a wide range of natural products, pharmaceuticals, finechemicals, and food ingredients.

Free enzymes, however, are not always ideal in practicalapplications because they are often unstable and easily dena-tured and therefore are unsuitable for use in organic solventsor at high temperatures. In addition, the isolation and produc-

tion of enzymes and their one-time usage as catalysts arecostly. However, these major deterrents may be eliminated bythe use of immobilized enzymes. Immobilization of enzymescan be achieved by methods of varied complexity and effi-ciency (2) on a variety of supports. For example, enzymes canbe adsorbed onto insoluble materials, copolymerized with areactive monomer, encapsulated in gels, cross-linked with abifunctional reagent, covalently bound to an insoluble carrier(3), or entrapped within an insoluble gel matrix of natural orsynthetic resin (2).

Hydrogels are polymeric materials made from hydrophilicand/or hydrophobic monomers, which can be a homopolymeror a copolymer. Their major characteristic is that they can im-bibe large quantities of water without dissolution of the poly-mer network. This feature makes them interesting supportsfor immobilization of enzymes. In addition to providing thewater needed for enzyme activity, the hydrogel also can ab-sorb water produced during the esterification reaction, thusincreasing the products.

In this work, a new method of immobilization of Candidarugosa lipase by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) hydrogel was carried out.The activities and characteristics of the immobilized lipasepreparations were investigated.

MATERIALS AND METHODS

Materials. Lipase from C. rugosa (Type VI); monomers N-vinyl-2-pyrrolidone (VP) and 2-hydroxyethylmethacrylate(HEMA); and cross-linker ethylene dimethacrylate (EDMA)were obtained from Sigma Chemical Co. (St. Louis, MO).Initiator, α,α′-azoisobutyronitrile (AIBN), was from FlukaChemical, Buchs, Switzerland. All other reagents were of an-alytical grade. The organic solvents and substrates were driedover molecular sieves (3Å) before use.

Purification of monomers.VP and HEMA were purified bypassing through an aluminum oxide column (2.5 × 10.0 cm)until colorless products were obtained. EDMA was used asreceived.

Preparation of lipase solution. Commercial lipase from C.rugosa (500 mg) was dispersed in distilled water (10.0 mL).

Copyright © 1999 by AOCS Press 571 JAOCS, Vol. 76, no. 5 (1999)

*To whom correspondence should be addressed at Jabatan Kimia, UniversitiPutra Malaysia, 43400 Serdang, Malaysia.E-mail: [email protected]

Immobilization of Lipase on Poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) Hydrogel

for the Synthesis of Butyl OleateM. Basri*, C.C. Wong, M.B. Ahmad, C.N.A. Razak, and A.B. Salleh

Center for Research in Enzyme & Microbial Technology, Fakulti Sains & Pengajian Alam Sekitar,Universiti Putra Malaysia, 43400 Serdang, Malaysia

This mixture was agitated on a vortex mixer, centrifuged at13,000 rpm for 10 min, and the supernatant used for lipaseimmobilization.

Lipase immobilization. Purified monomers, VP andHEMA, of varying weight percentage (wt%) compositionwere mixed together with 1% EDMA (wt%) in a clean, dryflask. The composition of hydrogels prepared wereVP(%)/HEMA(%) [% = wt% of monomer in total weight of(VP + HEMA)]: 0:100, 10:90, 30:70, 50:50, 70:30, 90:10 and100:0. To these mixtures, dry initiator, α,α′-azoisobutyroni-trile (AIBN) (10−4 moles) was added and the flasks shakenuntil the AIBN dissolved. The mixtures were then transferredto a polymerization tube and the solutions degassed with ni-trogen for 15 min to remove oxygen. The mixtures were in-cubated to polymerize in a 55–60°C water bath. After thepolymer solutions became viscous (1–4 h), the polymers werecooled to 50°C, and lipase solution (1.0 mL), previously de-gassed with nitrogen, was added and the polymer solutionsshaken until homogeneous solutions were obtained. The so-lutions in the polymerization tubes were sealed with rubberstoppers and further polymerized at 50°C for about 5 h. Thesolid polymerized rods were removed from the polymeriza-tion tubes, cut into small pieces (0.2–0.4 cm3), and stored at−4°C prior to use.

Protein assay. Protein content of the hydrogels was deter-mined by using the method of Bradford (4) with bovine serumalbumin as standard. For the blank determination, poly(VP-co-HEMA) hydrogel without lipase was used.

Lipase activity. The assay system consisted of poly(VP-co-HEMA)-immobilized lipase (0.3 g), oleic acid (2.0 mmol),butanol (8.0 mmol), and hexane (2.6 mL). The mixture wasincubated at 37°C for 5 h in a horizontal water bath shaker at150 rpm. The reaction was terminated by dilution with ace-tone/ethanol (1:1 vol/vol, 3.5 mL). The residual free fatty acidin the reaction mixture was determined by titration withNaOH (0.2 M) using an automatic titrator (ABU 90, Ra-diometer, Copenhagen, Denmark) to pH 9.5. For the blankdetermination, poly(VP-co-HEMA) hydrogel without lipasewas used. The specific activity of the enzyme was expressedin µmol of free fatty acid used min−1 (mg protein)−1.

Gas chromatography. Reaction products were analyzedperiodically on a Shimadzu 8A gas chromatograph (Kyoto,Japan) using a 30 m polar capillary column Nukol TM (0.32mm, i.d.) from Supelco Inc. (Australia). Nitrogen was used ascarrier gas, at 1.0 mL/min. The injector and detector tempera-tures were set at 250°C. The initial column temperature was110°C. The temperature was increased at a rate of 8°C min to200°C.

Effect of monomers, cross-linker, and the poly(VP-co-HEMA) hydrogels on the activity of free lipase. The purifiedmonomers and monomer mixtures (0.5 mL), crosslinker(EDMA) (0.2 mL), and the poly(VP-co-HEMA) hydrogels(0.3 g) were placed into separate vials that contained the en-zyme assay solution and the native lipase [0.02–0.05 mg (pro-tein equivalent in 0.3 g immobilized lipase)]. The vials werethen incubated at 37°C in a horizontal water bath shaker at

150 rpm for 5 h. The enzyme activities are expressed as a per-centage of the activity compared to free lipase.

Lipase leaching. The poly(VP-co-HEMA)-immobilized li-pases (0.3 g) were placed into sealed vials with hexane (4.0mL). The mixtures were shaken at 30°C for 0.5 h in a horizon-tal water bath shaker at 150 rpm. The immobilized lipases wereisolated from the organic solvent by filtration through What-man No. 1 filter paper (one cycle). The above procedure wasrepeated accordingly up to four cycles after which residual en-zyme activities were determined. Activities are expressed as apercentage of the untreated immobilized preparations.

Thermostability of immobilized lipase. The poly(VP-co-HEMA)-immobilized lipases (0.3 g) were incubated in hexaneat various temperatures for 1 h in sealed vials. After incubation,the enzyme mixtures were cooled to room temperature and li-pase activity was determined at 37°C. The relative activities areexpressed as a percentage of the untreated immobilized lipase.

The stability of immobilized lipase in hexane at 40, 50, 60,and 70°C, with respect to incubation time was also investi-gated. The residual activities are expressed as a percentage ofthe enzyme activity at zero time.

Stability in organic solvent. The immobilized enzymeswere incubated in hexane for between 1 and 12 d at room tem-perature. Their residual activities were determined at 37°C.The residual activities are expressed as a percentage of theimmobilized lipase activity at day 0.

Storage stability of the immobilized lipase. The immobi-lized enzyme preparations were stored at room temperature(27–28°C), 4, 0, and −80°C for 30 d in sealed vials. Afterwarming preparations to room temperature, the residual ac-tivities were determined. The residual activities are expressedas a percentage of the immobilized lipase activity at day 0.

RESULTS AND DISCUSSION

Effect of monomers, crosslinker, monomer mixtures, andpoly(VP-co-HEMA) hydrogel polymers on lipase activity. Theeffect of monomers, crosslinker, and the poly(VP-co-HEMA)hydrogel on the esterification reaction of lipases is shown inFigure 1. Hydrogels of varying composition did not affect theactivity of lipase as shown. However, the presence of unre-acted monomers, VP and HEMA, in solution form, decreasedthe activity of the lipase to less than 50%. In contrast, thecross-linking agent EDMA gave 98% of the residual activity.The effect of the monomer mixtures on the enzyme at 50°Cwas studied since this was the temperature at which the en-zyme was introduced to the monomer mixtures. The resultsshowed that in the presence of hydrogels the enzyme retainedmore than 90% of the activity. Apparently, the reactivemonomers may have a poisoning effect on the enzyme, thusdecreasing its activity. However, if all the monomers are com-pletely polymerized, the gel has no effect on lipase activity.

Activity of immobilized lipases. The esterification resultsusing the poly(VP-co-HEMA)-immobilized lipase are shownin Figure 2. As expected, immobilization of the lipase ontohydrogels showed increased esterification activity compared

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FIG. 1. Effect of monomers, cross-linker, monomer mixtures, and poly(VP-co-HEMA) hydrogelon the activity of lipase. NL, native lipase; VP(%)/HEMA(%), 0:100, 10:90, 30:70, 50:50,70:30, 90:10 and 100:0, hydrogels with the respective compositions; VP(%)/HEMA(%), 0:100,10:90, 30:70, 50:50, 70:30, 90:10, and 100:0 (viscous), monomer mixtures (50°C) with the re-spective compositions; EDMA, ethylene dimethacrylate; VP, N-vinyl-2-pyrrolidone; HEMA, 2-hydroxyethylmethacrylate. [% = wt% of monomer in total weight of (VP + HEMA)].

FIG. 2. Esterification activities of poly(VP-co-HEMA)-immobilized lipases. VP(%)/HEMA(%),0:100, 10:90, 30:70, 50:50, 70:30, 90:10, and 100:0, hydrogels with the respective composi-tions. For other abbreviations see Figure 1.

to native lipase, except for the lipase entrapped on 100%HEMA, which showed a decrease in activity. Increasing thepercentage VP from 0 to 90, which corresponds to an increasein hydrophilicity of the hydrogel and its equilibrium watercontent (EWC) (5), seemed to increase the activity of the im-mobilized enzyme. Lipase immobilized on VP/HEMA hydro-gel, 90:10, is the best for the esterification reaction as it gavethe highest activity. This may be due to the available watersurrounding the enzyme in the hydrogel. Sufficient water isneeded to maintain the three-dimensional conformation of thelipase to retain active catalysis. Alternatively, the hydrophilicsupport may absorb the water produced in the esterificationreaction. This would favor the formation of products, result-ing in higher yields. The data obtained are in agreement withthe results of Kosugi and Suzuki (6), who reported that theactivity of an entrapped lipase depends on a high concentra-tion of water surrounding the catalytic surface of the lipaseusing high hydrophilic supports.

The low activity observed for lipase immobilized on 100%HEMA may be due to the decreased EWC of the HEMApolymer. The composition of the monomers in gel formationwas important to ensure sufficient water within the matrix.The lipase activity for the 100% HEMA polymer preparation

was relatively lower than the activity of lipase on VP/HEMA,90:10. This may be due to the partial solubility of this hydro-gel in water and the lower level of cross-linking, which re-sults in more lipase diffusing out of the gel.

Leaching study in hexane. The effect of hexane washingon lipase activity is shown in Figure 3. Increasing the hy-drophobicity of the hydrogel (increasing the percentageHEMA) decreased the leaching effect with hexane. Lipaseimmobilized on 100% HEMA hydrogel retained its activityduring the washing process, whereas lipase immobilized on100% VP exhibited the lowest retention stability in this ex-periment. As the HEMA content decreased, physical cross-linking (hydrophobic bonding) may be decreased, thus low-ering the entrapment stability.

Thermostability of the immobilized lipase. Immobilizationof lipase onto hydrogel seemed to increase its thermal stabil-ity compared to free lipase after 1 h incubation (Fig. 4). Theimmobilized preparations were more thermostable over thetemperature range of 40–70°C than the native lipase. The rel-ative activity of the immobilized lipase decreased starting at50°C with further decrease at 70°C. As the temperature israised, the EWC of the hydrogel gradually decreases, whichis accompanied by the shrinkage of gel matrix. This reduced

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FIG. 3. Leaching study of poly(VP-co-HEMA)-immobilized lipases by hexane washing.VP(%)/HEMA(%), 0:100, 10:90, 30:70, 50:50, 70:30, 90:10 and 100:0, hydrogels with the re-spective compositions. For abbreviations see Figure 1.

pore size in the hydrogel results in a decrease in the diffusionof substrate (7), thus resulting in a decrease in esterification.The decreased activity of the immobilized lipase at highertemperature also may be due to decreased hydrophobic inter-actions between the lipase and hydrogel.

The lipase, immobilized on VP/HEMA, 50:50 exhibitedthe highest resistance to thermal denaturation. The balance inhydrophobic and hydrophilic sites in this hydrogel seemed tostabilize the enzyme to heat-induced denaturation. The hy-drophilic sites (VP) offer the advantages of high water con-tent and softness, whereas the hydrophobic sites (HEMA)give rigidity and toughness to the hydrogel. In addition, thebalance in structure in this hydrogel may provide the rightamount of water for the lipase to function efficiently. Hydro-gels whose percentage composition varied from VP/HEMA,50:50, showed lower stability. The decrease in activity of im-mobilized lipase with respect to temperature may also be dueto the nonhomogeneous cross-linking of the polymer networknoted at high contents of VP.

Figure 5 shows the residual activity of lipase-immobilizedhydrogel of composition VP/HEMA, 50:50, incubated at var-ious temperatures with respect to time. Incubation at 40°C for3 h did not decrease the activity of the immobilized lipase

whereas at 50, 60, and 70°C incubation for 3 h decreased therelative activity of the immobilized lipase. Increasing the in-cubation time at higher temperature also changed the appear-ance of the gel, indicating that water may be evaporating fromthe matrix. Cantarella et al. (8) found that a hydrophilic ma-trix protects enzymes against thermal and chemical deactiva-tion compared to a hydrophobic matrix.

Stability in organic solvent. The stability of the immobi-lized lipase preparations in hexane were also investigated(Fig. 6). Lipase immobilized on VP/HEMA, 30:70, 50:50,and 70:30, retained their activities for the first 3 d, whereasonly the immobilized lipase on VP/HEMA, 50:50, retainedits activity after 8 d. The immobilized lipase on VP/HEMA,50:50, exhibited the highest half-life (11.50 d), whereas li-pase immobilized on VP/HEMA of other compositions andnative lipase had lower solvent stability. The native lipase ex-hibited the least stability in organic solvent indicating that ac-tive protein structure was denatured by the organic solvent.The solvent stability of lipase immobilized on VP/HEMA,0:100, 10:90, 90:10, and 100:0, were low. Hydrogels of highhydrophobicity and high hydrophilicity seemed to be least ef-fective in protecting the enzymes from unfavorable contactwith the organic solvent

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FIG. 4. Thermostability of poly(VP-co-HEMA) immobilized lipases incubated for 1 h in hexane.VP(%):HEMA(%), 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, and 100:0, hydrogels with the re-spective compositions. For abbreviations see Figure 1.

Storage stability of the immobilized lipase. The stability ofthe various immobilized lipases incubated in hexane for 30 dunder different storage conditions is shown in Table 1. All im-mobilized lipase preparations and the native lipase showed fullcatalytic activity after storage at −80°C. Immobilized lipasesretained their full activity when stored at 0°C, whereas the na-tive lipase showed 30% of activity. At 4°C, the lipase immo-bilized on the more hydrophobic hydrogels showed an in-

creased storage stability compared to the native lipase, withthe lipase immobilized on VP/HEMA hydrogel 50:50 havingthe best stability. However, lipase immobilized on the morehydrophilic hydrogels showed lower stability even when com-pared to native lipase. When stored at room temperature, theimmobilized lipases, except VP/HEMA 50:50, had decreasedstorage stability compared to native lipase (34% of activity).As observed in thermal treatment, lipase immobilized on hy-

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FIG. 5. Thermostability of poly(VP-co-HEMA) immobilized lipases (VP/HEMA, 50:50) with re-spect to time at 40, 50, 60, and 70°C. For abbreviations see Figure 1.

TABLE 1The Effect of Storage Condition on the Esterification Reaction by ImmobilizedLipase After 30 Days

Relative activity (%)a

Immobilized lipase RT 4°C 0°C −80°C

VP(%)/HEMA(%), 0:100 7 79 100 100VP(%)/HEMA(%), 10:90 8 80 100 100VP(%)/HEMA(%), 30:70 33 83 100 100VP(%)/HEMA(%), 50:50 63 90 100 100VP(%)/HEMA(%), 70:30 15 30 100 100VP(%)/HEMA(%), 90:10 3 21 100 100VP(%)/HEMA(%), 100:0 15 72 100 100Native lipase 34 50 67 100aActivity is expressed as percentage of the lipase activity at day 0. The ester synthesis is followed bythe rate of disappearance of oleic acid from the reaction mixture containing butanol and oleic acid.VP, N-vinyl-2-pyrrolidone; HEMA, 2-hydroxyethylmethacrylate; RT, room temperature.

drogel with the composition VP/HEMA 50:50 also exhibitedthe highest storage stability. The decrease in the relative activ-ity of the immobilized lipases of other compositions as com-pared to native lipase at room temperature may be attributedto the presence of water in the immobilized lipases introducedduring the immobilization procedure (9).

Our results indicate that immobilization of lipase on hy-drogels may be suitable for industrial applications. Their in-creased activity in organic solvents could lead to their use inthe increased esterification of fats and oils. Such increasedstability is favorable for commercial applications. The sim-plicity of the technique suggests that it may have wide usagefor biologically active proteins that stimulate their use in in-dustrial processes.

ACKNOWLEDGMENT

This project was financed by the Ministry of Science, Technologyand Environment, Malaysia.

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Amundson, Immobilized Lipase Reactors for Modification ofFats and Oils—A Review, J. Am. Oil Chem. Soc. 67:890–910(1990).

3. Stark, M.B., and K. Holmberg, Covalent Immobilization of Li-pase in Organic Solvents, Biotechnol. Bioeng. 34:942–950(1989).

4. Bradford, M.M., A Rapid and Sensitive Method for the Quanti-tation of Microgram Quantities of Protein Utilizing the Princi-ple of Protein–Dye Binding, Anal. Biochem. 72:248 (1976).

5. Mohamed, D., The Effect of Varying Copolymer Compositionon the Properties of Hydrogel, B.Sc. Thesis, University PutraMalaysia (1996).

6. Kosugi, Y., and H. Suzuki, Functional Immobilization of LipaseEliminating Lipolysis Product Inhibition, Biotechnol. Bioengin.40:346–374 (1992).

7. Park, T.G., Stabilization of Enzyme Immobilized in Tempera-ture-Sensitive Hydrogels, Biotech. Lett. 15:57–60 (1993).

8. Cantarella, L., F. Alfani, and M. Cantarella, Stability and Activ-ity of Immobilized Hydrolytic Enzymes in Two-Liquid-PhaseSystems: Acid Phosphatase, β-Glucosidase, and β-Fructofura-nosidase Entrapped in Poly(2-hydroxyethyl methacrylate) Ma-trices, Enz. Microb. Technol. 15:861–867 (1993).

9. Basri, M., K. Ampon, W.M.Z. Wan Yunus, C.N.A. Razak, andA.B. Salleh, Enzymic Synthesis of Fatty Esters by HydrophobicLipase Derivatives Immobilized on Organic Polymer Beads, J.Am. Oil Chem. Soc. 72:407–411 (1995).

[Received August 12, 1998; accepted January 5, 1999]

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FIG. 6. Stability of poly(VP-co-HEMA) immobilized lipases incubated in hexane for 12 days atroom temperature. NL, native lipase; VP(%):HEMA(%), 0:100, 10:90, 30:70, 50:50, 70:30,90:10, and 100:0, hydrogels with the respective compositions.